Bulletin of Pharmaceutical Research 2012;2(2):50-5 An Official Publication of Association of Pharmacy Professionals ISSN: 2249-6041 (Print); ISSN: 2249-9245 (Online)
RESEARCH ARTICLE
VALIDATED RP-HPLC METHOD FOR THE ESTIMATION OF TELMISARTAN IN TABLET FORMULATION M.V. Basaveswara Rao1*, A.V.D. Nagendrakumar2, M. Sivanadh3 and G. Venkata Rao4 1
Department of Chemistry and Pharmacy, Krishna University, Machilipatnam-521 001, Andhra Pradesh, India Department of Chemistry, GITAM University, Visakhapatnam-530 045, Andhra Pradesh, India 3 Department of Chemistry, ANR College, Gudivada-521 301, Andhra Pradesh, India 4 Department of Bio-Chemistry, SRR and CVR Govt. Degree College, Vijayawada-520 001, Andhra Pradesh, India 2
*E-mails:
[email protected],
[email protected] Tel.: +91-9346234562, +91-9700813924. Received: January 15, 2012 / Revised: May 10, 2012 / Accepted: May 11, 2012
A simple, specific, accurate and precise reverse phase high performance liquid chromatographic method is developed and validated for the estimation of telmisartan in tablet dosage form. The expected separation and peak shapes were obtained on chromosil C18 (250 mm x 4.6 mm, 5 µm) column. To have an ideal separation of the drug under isocratic conditions, mixtures of solvents like methanol, water and acetonitrile with or without different buffers in different combinations were tested as mobile phases on a chromosil C18 column. A mixture of methanol : 0.1% orthophosphoric acid : acetonitrile in the ratio of 80:05:15 v/v/v was proved to be the most suitable of all the combinations since the chromatographic peak obtained was better defined and resolved and was almost free from tailing. The flow rate was 1.5 ml/min and effluents were monitored at 256 nm. The retention time for telmisartan was 2.7 min. The method was validated and found to be accurate, and precise. Recovery of telmisartan from tablet formulation was found to be 99.41%. The proposed method was successfully applied for the quantitative determination of telmisartan in tablet formulation. Key words: Telmisartan, HPLC, UV detection, Validation, Methanol, Acetonitrile. INTRODUCTION Telmisartan is 2-(4-{[4-methyl-6-(1-methyl-1H1,3-benzodiazol-2-yl)-2-propyl-1H -1,3-benzodiazol-1-yl] methyl} phenyl) benzoic acid, with molecular formula C33H30N4O2 (Figure 1). It is a member of a family of drugs called angiotensin receptor blockers (ARBs), which include losartan (Cozaar), valsartan (Diovan), irbesartan (Avapro), candesartan (Atacand) and is used alone or in combination with other medications to treat high blood pressure (hypertension). High blood pressure reduction helps to prevent strokes, heart attacks, and kidney problems. It has the longest half-life of any ARB (24 h) and the largest volume of distribution. In addition to blocking the RAs, telmisartan acts as a selective modulator of peroxisome proliferato-activated
receptor gamma (PPAR-γ), a central regulator of insulin and glucose metabolism. Telmisartan’s dual mode of action may provide protective benefits against the vascular and renal damage caused by diabetes and cardiovascular disease (Benson et al 2004; PubMed Health). For the determination of telmisartan in pure and pharmaceutical dosage forms and in biological samples, the chromatographic conditions comprised of a reversed phase C8 column (4.6 ´ 150 mm, 3.5 m) with mobile phase composed of buffer and methanol (40 : 60 v/v, adjusted the pH to 3.0 with ortho phosphoric acid). Selected mobile phase was a combination of acetonitrile:buffer (0.01 M potassium dihydrogen phosphate) in ratio 65:35, pH 4.0 adjusted with
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Bull. Pharm. Res. 2012;2(2) for investigation. The chromatographic analysis was performed on a Chromosil C18 column (250 mm × 4.6 mm, 5 µm). Degassing of the mobile phase was done using a Loba ultrasonic bath sonicator. A Denwar analytical balance was used for weighing the materials. Chemicals and solvents The reference sample of Telmisartan (API) was obtained from Cipla, Mumbai. The Formulation CRESAR (Telmisartan) was procured from the local market. Methanol, acetonitrile used were of HPLC grade and purchased from Merck Specialities Private Limited, Mumbai, India. Orthophosphoric acid used was AR grade purchased from local market. The mobile phase A mixture of methanol : 0.1% orthophosphoric acid : acetonitrile in the ratio of 40:50:10 v/v/v was prepared and used as mobile phase.
Fig. 1. Structure of telmisartan orthophosphoric acid. The method was carried out in TLC precoated silica gel on aluminum plate 60 F 254, (10 cm ×10 cm, prewashed by methanol and activated at 60° C for 5 min prior to chromatography). A Genesis C18 column having dimensions of 4.6 × 250 mm and particle size of 5 µm in isocratic mode, with mobile phase containing a mixture of 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 using orthophosphoric acid) : methanol : acetonitrile (15:15:70 v/v/v) was used. Chromatography was performed on a ODS Hypersil C18 (25 cm × 4.6 mm ID) column from thermo in isocratic mode with mobile phase containing acetonitrile : 0.05 M KH2PO4 (pH 3.0) (60:40). Literature is enriched with several scientific reports indicating availability of UV and RPHPLC methods for determination of drugs (Kurade et al 2009; Sujana et al 2011; Kottai Muthu et al 2011; Prasanthi et al 2011; Bhimavarapu et al 2011) but yet no method is reported for the estimation of telmisartan in tablet formulation. So, in continuation of our work on developing new RP-HPLC methods for determination of drugs (Basaveswara Rao et al 2012), an attempt was made to develop and validate a new RP-HPLC method for the estimation of telmisartan in tablet formulation.
The buffer and standard solution of the drug About 1.0 ml of orthophosphoric acid was diluted to 1000 ml with water and filtered through 0.45 µ nylon filter. For analysis, 100 ppm standard solution was prepared and required concentrations were obtained from the 100 ppm solution by appropriate dilutions. Sample (tablet) solution The formulation tablets of telmisartan (CRESAR 20 mg) were crushed to give finely powdered material. From the powder, 12 ppm solution was prepared in mobile phase and then filtered through Ultipor N66 Nylon 6, 6 membrane sample filter paper. Method development For developing the method (ICH Q2A, 1995; ICH Q2B, 1997; Shabir, 2003; USP 30, 2007), a systematic study of the effect of various factors was undertaken by varying one parameter at a time and keeping all other conditions constant. Method development consists of selecting the appropriate wave length and choosing stationary and mobile phases. The following studies were conducted for this purpose: Detection of wavelength The spectrum of 10 ppm solution of telmisartan in methanol was recorded separately on UV spectrophotometer. The peak of maximum absorbance wavelength was observed.
EXPERIMENTAL Instrumentation Peak HPLC containing LC 20AT pump and variable wavelength programmable UV-Visible detector and Rheodyne injector was employed
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Choice of stationary phase and mobile phase Finally the expected separation and peak shapes were obtained on chromosil C18 (250 mm ´ 4.6 mm, 5 µm) column.
was calculated by comparing the area before and after the addition of the standard drug. The standard addition method was performed at 50%, 100% and 150% level of 12 ppm. The solutions were analyzed in triplicate at each level as per the proposed method. The percent recovery and % RSD was calculated.
Flow rate Flow rates of the mobile phase were changed from 0.5-1.5 ml/min for optimum separation. It was found from experiments that 1.5 ml/min flow rate was ideal for elution of analyte.
Robustness The robustness study was performed by slight modification in flow rate of mobile phase, pH of the buffer and composition of the mobile phase. Telmisartan at 6 ppm concentration was analyzed under these changed experimental conditions.
Optimization of chromatographic conditions Chromatographic conditions are required to be optimized. These optimized conditions were followed for the determination of telmisartan in bulk samples and in its formulations.
System suitability The system suitability was studied under each validation parameters by injecting the six replicates of the standard solution (2 ppm).
Validation of proposed method The proposed method was validated as per ICH guidelines (Hokanson, 1994). The parameters studied for validation were specificity, linearity, precision, accuracy, robustness, system suitability, limit of detection, limit of quantification and solution stability.
Limit of detection and Limit of quantification Limit of detection (LOD) is the lowest concentration of analyte that gives a detectable response whereas limit of quantification (LOQ) is the lowest concentration that can be quantified reliably with a specified level of accuracy and precision. For this, sample was dissolved by using mobile phase and injected until peak was disappeared.
Specificity The specificity of method was performed by comparing chromatograms of blank, standard and sample (prepared from formulation). Linearity Linearity was performed by preparing mixed standard solutions of telmisartan at different concentration levels including working concentration mentioned in experimental condition i.e. 12 ppm. The response was read and the corresponding chromatograms were recorded. From these chromatograms, the mean peak areas were calculated and linearity plots of concentration over the mean peak areas were constructed individually.
Formulation The proposed method was applied to the assay of commercial tablets containing telmisartan. Sample was analyzed for five times after extracting the drug as mentioned in sample preparation for assay as described in the experimental section. RESULTS AND DISCUSSION To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry was found in a mixture of acetonitrile : methanol : 0.1% orthophosphoric acid in the ratio of 80:15:05 v/v and 1.5 ml/min flow rate proved to be better than the other mixtures in terms of resolution and peak shape (Table 1). The chromatogram of standard (4 ppm) is shown in the Figure 2. The optimum wavelength for detection was set at 256 nm at which much better detector responses for drug was obtained. The regressions of the plots were computed by least
Intraday and interday precision Precision of the method was performed as intraday precision and interday precision. To study the intraday precision, six replicate standard solution (12 ppm) of telmisartan was injected. The percent relative standard deviation (% RSD) was calculated. Accuracy The accuracy of the method was determined by standard addition method. A known amount of standard drug was added to the fixed amount of pre-analyzed tablet solution. Percent recovery
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Table 1. Optimized chromatographic conditions for estimation of telmisartan Mobile phase Pump mode Mobile phase pH Diluent Column Column temperature Wavelength Injection volume Flow rate Run time Retention time
MeOH : 0.1 % OPA : ACN :: 80:05:15 v/v/v Isocratic 5.8 Mobile phase Chromosil C18 column (250 mm ´ 4.6 mm, 5 µ) Ambient 256 nm 20 µl 1.5 ml/min 6 min 2.7 min
and slope (45459.12). The results obtained were within acceptable limits where capacity factor >2.0, tailing factor ≤2.0 and theoretical plates >2000. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. The retention times were 2.7 min for telmisartan. The number of theoretical plates was found to be 6638.20, which indicated efficient performance of the column. It was found that there was no interference due to excipients in the tablet formulation and also showed good correlation between the retention times of standard and sample. The specificity results suggested no peak for blank and retention time 2.790 min for telmisartan. Results of system precision studies are shown in Table 2 and Table 3. The R.S.D. for intraday precision studies was found to be 0.039 and that of interday precision was 0.123, which were well within the acceptable criteria of not more than 2.0. Low values of standard deviation denoted very good repeatability of the measurement. For the interday precision, a study carried out on consecutive days indicated good method precision. Standard addition method at 50%, 100% and 150% to the proposed HPLC method was carried out to find the accuracy of the telmisartan. The results showed good recoveries ranging from 99.00 to 101.45%. The mean recovery data obtained for each level as well as for all levels combined were within 2.0% of the label claim for the active substance with an R.S.D.
